GENETIC REVERSION AND NMR AS PROBES OF PROTEIN STRUCTURE

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Sequences and structures for many proteins are known, but the relationship between these properties is not well understood. A common protein structural motif is the interaction between alpha-helices. Elucidating rules governing such interactions is a prerequisite to the goal of protein engineering--de novo design of protein structure and function. When protein design is possible, new, complex drugs may be synthesized using chemistry common to biology, but problematic to synthetic organic chemistry. Design would also allow improvement of therapeutic proteins such as TPA. My research plan is to search for rules governing alpha-helical interaction in the protein, cytochrome c, using the technique of random, intragenic, second-site reversion (RISR) mutagenesis to alter helices, and the powerful new technique, 2-dimensional nuclear magnetic resonance (2-D NMR), to determine the solution structure of the altered proteins. Unlike site-directed mutagenesis, random mutagenesis makes few assumptions about the role of single residues and allows the system to tell the investigator which elements of sequence are important to structure. However, most random mutations are silent. RISR mutagenesis, which is a two stage process, eliminates silent, random mutations. First, random mutations are produced which disrupt gene function. Then a second set of random mutations is introduced at a different site, which resurrect function. The specific aim of the proposed research is to generate RISR mutations by separate insertion of random oligonucleotide cassettes spanning 4 contiguous codons into the N- and C-terminal alpha-helices of cytochrome c. The solution structure of second-site revertants, as determined using 2-D NMR, will lead to proposals concerning sequence requirements for helix interactions. These proposals will then be tested by making site-directed mutants. The yeast cytochrome c system, which I have worked with for 5 years, possesses the features necessary to produce RISR mutants.